Elsevier

Biomaterials

Volume 31, Issue 30, October 2010, Pages 7726-7737
Biomaterials

Plastic compressed collagen as a biomimetic substrate for human limbal epithelial cell culture

https://doi.org/10.1016/j.biomaterials.2010.07.012Get rights and content

Abstract

We describe, for the first time, the use of cellular plastic compressed collagen as a substrate for human limbal epithelial cell expansion and stratification. The characteristics of expanded limbal epithelial cells on either acellular collagen constructs or those containing human limbal fibroblasts were compared to a human central cornea control. After compression, human fibroblasts in collagen constructs remained viable and limbal epithelial cells were successfully expanded on the surface. After airlifting, a multilayered epithelium formed with epithelial cell morphology very similar to that of cells in the central cornea. Immunochemical staining revealed expression of basement membrane proteins and differentiated epithelial cell markers found in native central cornea. Ultrastructural analysis revealed cells on collagen constructs had many features similar to central cornea, including polygonal, tightly opposed surface epithelial cells with microvilli and numerous desmosomes at cell–cell junctions. Taken together, these data demonstrate that plastic compressed collagen constructs can form the basis of a biomimetic tissue model for in vitro testing and could potentially provide a suitable alternative to amniotic membrane as a substrate for limbal epithelial cell transplantation.

Introduction

The limbal epithelial stem cell (LESC) population of the cornea is responsible for maintaining the integrity of the outer epithelial surface. Destruction of this population can be caused by thermal or chemical injury, Stevens-Johnson syndrome, multiple surgeries, contact lens wear or microbial infection. This can lead to absence of an intact epithelial layer, conjunctival ingrowth, corneal neovascularisation, chronic inflammation and discomfort and ultimately, impaired vision [1].

One current treatment for LESC deficiency is autologous transplantation of cultured limbal epithelial stem cells from the uninjured eye or allogeneic transplant from a cadaveric or living relative donor. In this process, cells are commonly cultured on an amniotic membrane (AM) substrate before transfer to the injured eye [2], [3], [4], [5], [6]. Amniotic membrane has many favourable qualities for use in this context including anti-inflammatory, anti-angiogenic and anti-scarring properties [7]. Unfortunately, there are a number of drawbacks associated with its use including lack of a reliable supply of membranes, considerable biological variation between donors, costly screening processes and a lack of optimal transparency, which is obviously a major issue when dealing with repair of the cornea. With this in mind, many attempts have been made to develop an alternative to AM as a substrate for LESCs for use in corneal repair (reviewed by Levis et al. [8]). An ideal alternative would allow cells to proliferate to confluence, differentiate, produce appropriate basement membrane products and allow suitable cell–cell and cell matrix interactions.

Collagen has a number of useful characteristics when used as a cellular substrate; it is biocompatible, has low immunogenicity, is naturally remodelled by cells and is relatively inexpensive to isolate [9]. Collagen hydrogels contain a large proportion of water and so are inherently weak but if subjected to a process of plastic compression the gel properties can be altered without the need for chemical crosslinking. Collagen hydrogels can be seeded directly with fibroblasts before compression, resulting in thin, optically clear, cellular collagen membranes which are mechanically strong [10].

Here we describe the use of plastic compressed (PC) collagen as a substrate for human limbal epithelial cell (LEC) expansion and stratification into a corneal epithelial equivalent.

Section snippets

Donor tissue

Cadaveric donor corneal rims with appropriate research consent were obtained from Moorfields Lions Eye Bank (UK). Ethical permission for this study was obtained from the Research Ethics Committee (UK) (Ref No. 08/H0715/83). Corneas were stored at 4 °C in Optisol (Chiron Ophthalmics Inc. Irvine, California) after enucleaction and prior to LEC and fibroblast isolation.

Isolation and culture of human limbal epithelial cells

Human donor corneal rims were cut into quarters and incubated with 1.2 U ml−1 dispase II (Roche Diagnostics GmbH, Mannheim,

Collagen constructs

Cellular collagen gels could be cast and compressed rapidly and simply to produce thin (100–150 μm), transparent constructs (Fig. 2A) with a collagen concentration that increased from 0.2% before compression to approximately 11%. Constructs were easy to handle in liquid and maintained sufficient mechanical strength to withstand manipulation on the surface of a porcine eye. The constructs were able to lie flat on the convex cornea surface with no evidence of folding or puckering and could be

Discussion

This study demonstrates that PC collagen can be used to support the expansion and stratification of human corneal epithelial cells. The resulting cellular collagen constructs display many of the typical characteristics of human corneal epithelium.

When attempting to engineer a corneal equivalent or develop an in vitro corneal model it must be taken into consideration that the substrate should be biocompatible, mechanically stable, optically transparent and allow cell adhesion, migration and

Conclusions

The human corneal epithelial constructs produced using PC collagen as a substrate can be easily standardized and replicated to the required specifications. The resulting multilayered epithelial has many characteristics of human central corneal epithelium, including typical epithelial marker and basement membrane marker expression as well as ultrastructural similarities, and so could easily form the basis of an in vitro model of the corneal epithelium. Furthermore, the plastic compressed

Conflict of interest

The authors have no conflict of interest.

Acknowledgements

We would like to thank Dr. Alex Shortt for his assistance with the transfer of collagen constructs to the porcine eye and Tijna Alekseeva for her assistance with scanning electron microscopy. This study was supported by the Technology Strategy Board, the EPSRC (HL) and the National Institute for Health Research Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital and UCL Institute of Ophthalmology (JTD).

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